TISSUE CULTURE

 TISSUE CULTURES

The term ‘plant tissue culture culture’ broadly refers to the in vitro cultivation of

plants, seeds, plant parts on nutrient media under aseptic conditions.

History of Tissue Culture

1902         Haberlandt First attempt of plant tissue culture (Father of Plant Tissue culture)

1904         Hannig First attempt to culture embryo of selected crucifers

1922         Knudson Asymbiotic germination of orchid seeds in vitro

1922         Robbins In vitro culture of root tips

1925         Laibach Use of embryo culture technique in interspecific crosses of linseed ( linum)

1934         Gautheret In vitro culture of the cambial tissue of a few trees and shrubs,

                 Althoughugh failed to sustain cell division.

1934         White Successful culture of tomato roots

1939         Gautheret, Nobecourt and White Successful establishment of continuously growing

                 callus cultures

1940         Gautheret In vitro culture of cambial tissues of Ulmus to study

                 adventitious shoot formation

1941         Van Overbeek Use of coconut milk containing a cell division factor

                 for the first time to culture Datura embryos

1941         Braun In vitro culture of crown gal tissues

1944         Skoog In vitro adventitious shoot formation in tobacco

1946         Ball Raising of whole plants of Lupinus and Tropaeolum by

                 shoot tip culture

1950         Ball Regeneration of organs from callus tissue of Sequoia

                 sempervirens

1952         Morel and Martin Use of meristem culture to obtain virus-free Dahlias

1952         Morel and Martin First application of micrografting

1953         Tulecke Production of haploid callus of the gymnosperm

                 Ginkgo biloba from pollen

1954         Muir et al First plant regenerated from a single cell

1955         Miller et al Discovery of kinetin, a cell division hormone

1956         A, Kornberg et al In vitro synthesis of DNA

1957         Skoog and Miller Discovery of the regulation of organ formation by

                 changing the ratio of auxin : cytokinin

1958         Maheshwari and Rangaswamy Regeneration of somatic embryos in vitro from the

                 nucellus of Citrus ovules

1959         Reinert and Steward Regeneration of embryos from callus clumps and cell

                 suspensions of carrot (Daucus carota )

1959         Gautheret Publication of first handbook on “Plant Tissue Culture”

1960         Kanta First successful test tube fertilization in papaver rhoeas

1960         E. Cocking Enzymatic degradation of cell walls to obtain large

                 number of protoplasts

1960        Bergmann Filtration of cell suspensions and isolation of single

                cells by plating

1962        Murashiqe and Skoog Development of Murashige and Skoog nutrition medium

1964        Guha and Maheshwari Production of first haplo id plants from pollen grains of    

                Datura (Anther culture)

1968        H.G. Khorana Awarded Nobel prize for deciphering of genetic code

                H.G. Khorana et al. Deduced the structure of a gene for yeast alanyl tRNA

1968        Meselson and Yuan Coined the term “Restriction endonuclease” to describe a class  

                Of enzymes  involved in cleaving DNA

1970        Carlson Selection of biochemical mutants in vitro by the use of

                tissue culture derived variation

1970        Power et al. First achievement of protoplast fusion

1970        H. Temin and D. Baltimore Discovered the presence of reverse transcriptase (a

                RNA directed DNA polymerase which has the ability to synthesize cDNA

                using      mRNA as a template

1970        Smith Discovery of first restriction endonuclease from Haemophillus influenzae Rd.

                It     was later purified and named Hind 11

1971        Nathans Preparation of first restriction map using Hind II enzyme to cut circular

                DNA    or SV 40 into 11 specific fragments

1971        Takebe et al, Regeneration of first plants from protoplasts

1972        Carlson et al, First report of interspecific hybridization through

                protoplast fusion in two species of Nicotiana

1972        Berg et al, First recombinant DNA molecule produced using

                restriction enzymes

1974        Reinhard Biotransformation in plant tissue cultures

1974        Zaenen et al. ; Larebeke

                et al.Discovered the fact that the Ti plasmid was the tumor

                inducing principle of Agrobacterium

1976        Seibert Shoot initiation from cryo-preserved shoot apices of carnation

1976        Power et al. Inter -specific hybridization by protoplast fusion or

                Petunia hydrida and P. parodii

1978        Melchers et al. Somatic hybridization of tomato and potato resulting in

                pomato

1979       Marton et al. Co-cultivation procedure developed for transformation

               of plant protoplasts with Agrobacterium

1984       De Block et al.; Horsch et al.

               Transformation of tobacco with Agrobacterium;

                transgenic plants developed

*************TYPES OF TISSUE CULTURES************

Plant Tissue Culture: Plant tissue culture, which covers all types of aseptic plant culture should be used as a restricted sense and it is possible to distinguish it into various types of cultures.

• Seed Culture : Culture of seeds in vitro to generate seedlings/plants

• Embryo Culture : Culture of isolated mature or immature embryos.

• Organ Culture: Culture of isolated plant organs. Different types can be DO, the distinguished, e.g. meristem, shoot tip, root culture, anther tissue culture

• Callus Culture: Culture of a differentiated tissue from explant allowed to dedifferentiate in vitro and a so-called callus tissue is produced.

• Cell culture: Culture of isolated cells or very small cell aggregates remaining dispersed in liquid medium

• Protoplast culture: Culture of plant protoplasts, i,e., cells devoid of their cell walls.

• Anther culture: Culture of anthers.

Seed culture

Seed culture is an important technique when explants are taken from in vitro-derived plants and in propagation of orchids. Sterilizing procedures are needed for plant materials that are to be used directly, as explant source can cause damage to tissues and affect regeneration. In that case, culture of seeds to raise sterile seedling is the best method. Orchid cloning in vivo is a very slow process. Thus seeds can be germinated in vitro and vegetatively propagated by meristem culture is then carried out on a large scale. Most orchids are sown in vitro because:

(i) orchid seeds are very small and contain very little or no food reserves. Their small size (1.0-2.0 mm long and 0.5-1,0 mm wide) makes it very likely that they can be lost if sown in vivo, and the limited food reserves also make survival in vivo unlikely. The seed consists of a

thickened testa, enclosing an embryo of about 100 cells. The embryo has a round or spherical form. Most orchid seeds are not differentiated: there are no cotyledons, roots and / or endosperm. The cells of an embryo have a simple structure and are poorly differentiated;

i) sowing in vitro makes it possible to germinate immature orchid embryos, thus shortening the breeding cycle; and

ii) germination and development take place much quicker in vitro since there is a conditioned environment and no competition with fungi or bacteria.

Orchid seeds imbibe water via the testa and becomes swollen. After cell division begun, the embryo cracks out of the seed coat. A protocorm-like structure is formed from the clump of cells and on this a shoot meristem can be distinguished. Protocorm has a morphological state that lies between an undifferentiated embryo a shoot. Protocorms obtained by seed germination have many close similarities with those produced from isolated shoot tips; the term protocorm like-bodies has introduced when cloning orchids by meristem culture. The vegetative propagation of orchids follows culture of seeds, transformation of meristem into protocorm-like bodies, the propagation of protocorms by cutting them into pieces and the development of these protocorms to rooted shoots.

A large number of factors influence the germination and growth of orchids. The mineral requirement of orchids is generally not high and a salt poor medium of Knudson (1946) and Vacin and Went (1949) are good. Some of the orchids require s (Paphiopedilum ciliolare) for germination while others require low irradiance. Sugar is extremely important as an energy source, especially for those that germinate in darkness. Regulators are usually not necessary for seed germination, and their addition often leads to unwanted effects like callus formation, adventitious shoot formation, etc.

 Embryo culture

Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant. The first attempt to grow the embryos of angiosperms was made by Hannig in 1904 who obtained viable plants from in vitro isolated embryos of two crucifers Cochleria and Raphanus (Hannig 1904). In 1924, Dietrich grew embryos of different plant species and established that mature embryos grew normally but those excised from immature seeds failed to achieve the organization of a mature embryo (Dietrich, 1924). They grew directly into seedlings, skipping the stages of normal embryogenesis and without the completion of dormancy period. Laibach (1925, 1929) demonstrated the practical application of this technique by isolating and growing the embryos of interspecific cross. Linum perenne and L, austriacum that aborted in vivo. This led Laibach to suggest that in all crosses where viable seeds are not formed, it may be appropriate to exercise their embryos and grow them in an artificial nutrient medium. Embryo

Culture is now a well-established branch of plant tissue culture.

There are two types of embryo culture:

i) Mature embryo culture: It is the culture of mature embryos derived from ripe seeds. This type of culture is done when embryos do not survive in vivo or become dormant for long periods of time or is done to eliminate the inhibition of seed germination. Seed dormancy of many species is due to chemical inhibitors or acids, mechanical resistance present in the structures covering the embryo, rather than dormancy of the embryonic tissue.

ii) Immature embryo culture/embryo rescue: It is the culture of immature embryos to rescue the embryos of wide crosses. This is mainly used to avoid embryo abortion with the purpose of producing a viable plant. The underlying principle of embryo rescue technique is the aseptic isolation of embryo and its transfer to a suitable medium for development under optimum culture conditions. Florets are removed at the proper time and either florets or ovaries are sterilized. Ovules can then be removed from the ovaries. The tissue within the ovule, in which the embryo is embedded, is already sterile. For mature embryo culture either single mature seeds are disinfected or if the seeds are still unripe then the still closed fruit is disinfected. The embryos can then be aseptically removed from the ovules. Utilization of embryo culture to overcome seed dormancy requires a different procedure. Seeds that have hard coats are sterilized and soaked in water for few hours to few days. Sterile seeds are then split and the embryos excised.

The most important aspect of embryo culture work is the selection of medium

necessary to sustain continued growth of the embryo. In most cases a standard basal plant growth medium with major salts and trace elements may be utilized.

TECHNIQUE:

Mature embryos can be grown in a basal salt medium with a carbon energy source such as sucrose. But young embryos in addition require different vitamins, amino acids, and growth regulators and in some cases natural endosperm extracts. Young embryos should be transferred to a medium with high sucrose concentration (8-12%); which approximate the high osmotic potential of the intracellular environment of the young embryosac, and a combination of hormones which supports the growth of heart-stage embryos (a moderate level of auxin and a low level of cytokinin).

Reduced organic nitrogen as aspargine, glutamine or casein hydrolysate is always beneficial for embryo culture. Malic acid is often added to the embryo culture medium. After one or two weeks when embryo ceases to grow, it must be transferred , to a second medium with a normal sucrose concentration, low level of auxin and a moderate level of cytokinin which allows for renewed embryo growth with direct shoot germination in many cases. In some cases where embryo does not show shoot formation directly, it can be transferred to a medium for callus induction followed by shoot induction. After the embryos have grown into plantlets in vitro, they are generally transferred to sterile soil and grown to maturity.

Applications of embryo culture

1. Prevention of embryo abortion in wide crosses: Successful interspecific hybrids have been seen in cotton, barley, tomato, rice, legume, flax and well known intergeneric hybrids

include wheat x barley, wheat x rye, barley x rye, maize x Tripsacum, Raphanus sativus x Brassica napus. Distant hybrids have also been obtained via embryo rescue in Carica and Citrus species. Embryo rescue technique has been successfully used for raising hybrid embryos between Actidinia

deliciosa x A. eriantha and A. deliciosa x A. arguata.

Resistance traits transferred to cultivated species through embryo rescue technique.

Crossing species Resistance trait(s)

Lycopersicon esculentum x L.peruvianum Virus, fungi and nematodes

Solanum melongena x S. khasianum Brinjal shoot and fruit borer

(Leucinodes arbonalis)

Solanum tuberosum x S. etuberosum Potato leaf roll virus

Triticum aestivum x Thynopyrum scripeum Salt tolerance

Hordeum sativumn x H. vulgare Powdery mildew and spot blotch

Hordeum vulgare x H. bulbosum Powdery mildew

Oryza sativa x O. minuta Blast (Pyricularia grisea) and Bacterial blight (Xanthomonas oryzae)

2. Production of Haploids: Embryo culture can be utilized in the production haploids or monoploids. Kasha and Kao (1970) have developed a technique to produce barley monoploids. Interspecific crosses are made with Horeum bulbosum as Lhe pollen parent, and the resulting hybrid embryos are cultured but they exhibit H. bulbosum chromosome elimination resulting in monoploids of the female parent H. vulgare.

3. Overcoming seed dormancy: Embryo culture technique is applied to break dormancy. Seed dormancy can be caused by numerous factors including endogenous inhibitors, specific light requirements, low temperature, storage requirements and embryo immaturity. These factors can be circumvented by embryo excision and culture.

4. Shortening of breeding cycle: There are many species that exhibit seed dorm that is often localized in the seed coat and/or in the endosperm. By removing these inhibitions, seeds germinate immediately. Seeds sometimes take up and O2 very slowly or not at all through the seed coat, and so germinate slowly if at all, e.g. Brussels sprouts, rose, apple, oil palm and iris. H (Ilex) are important plants for Christmas decorations. Ilex embryos remain in the immature heart-shaped stage though the fruits have reached maturity.

5. Prevention of embryo abortion with early ripening stone fruits: Some species produce sterile seeds that will not germinate under appropriate conditions and eventually decay in soil e.g. early ripening varieties of peach, cherry, apple, plum. Seed sterility may be due to incomplete embryo development, which results in the death of the germinating embryo. In crosses of early ripening stone fruits, the transport of water and nutrients to the yet immature

e is sometimes cut off too soon resulting in abortion of the embryo. Eg: Macapuno coconuts are priced for their characteristic soft endosperm which fills the whole nut. These nuts always fail to germinate because the endosperm invariably rots before germinating embryo comes out of the shell. Embryo culture has been practised as a general method in horticultural crops include avocado, peach, nectarine and plum. Two cultivars 'Goldcrest peach’ and 'Mayfire nectarine' have resulted from embryo culture and commercially grown.

6, Embryos are excellent materials for in vitro clonal propagation. This is especially true for conifers and members of Gramineae family.

7. Germination of seeds of obligatory parasites without the host is impossible in vivo, but is achievable with embryo culture.